Low-pass genome sequencing
Genomic DNA was extracted from prenatal specimens according to the manufacturer’s protocol. An STR-based semi quantitative PCR assay was used to check for maternal DNA contamination from this procedure. Samples with >10% contamination were excluded from the study. Finally, 200 ng of genomic DNA was randomly fragmented, and DNA libraries were constructed by end-repaired, A-tailed, and adaptor ligation.28
Low-pass GS was performed as previously described, with a mean coverage of 0.06X.5, 28, 29 Mapped reads were allocated to 20-kilobase (kb) bin sizes with 5-kb sliding to identify CNVs. CNV profiles of each chromosome were represented as log2 of the mean sequencing reads of each sequencing bin along the chromosome. Any two CNVs with ≥ 60% reciprocal overlap were identified as the same. Publicly available genomic databases including 1000 genomes, DGV (http://dgv.tcag.ca/dgv/app/home), OMIM (https://www.omim.org/), DECIPHER (https://decipher.sanger.ac.uk/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), UCSC (http://genome.ucsc.edu/), and PubMed (https://pubmed.ncbi.nlm.nih.gov/) were used as reference CNV sources. The pathogenicity of identified CNVs was assessed based on the American College of Medical Genetics (ACMG) guidelines.30 Only germline aneuploidy and pCNV were considered in further analyses.